The objective of this grant is to define at the cellular and molecular levels how the steroid hormone 1alpha,25 dihydroxyvitamin D3 [1a,25(OH)2D3] produces its biological responses. 1a,25(OH)2D3 is an unusually conformationally flexible molecule and consequently generates a wide array of molecular shapes that are available for binding to receptors. 1a,25(OH)2D3 generates biological responses via activation of at least two signal transduction pathways: (a) one shape of 1a,25(OH)2D3 interacts with a nuclear receptor [VDRnuc] to form a competent ligand-receptor partnership that, with other nuclear proteins, creates a transcription complex that regulates mRNA coding for selected proteins; and (b) a different shape of 1a,25(OH)2D3 interacts with a putative membrane [VDRmem] receptor to stimulate signal transduction events to activate raid responses [opening of Ca2+ and Cl- channels; activation of MAP-kinase; stimulation of intestinal Ca2+ transport or transcaltachia]. Functional analyses show that conformationally restricted analogs of 1 a,25(OH)2D3 VDRnuc and VDRmem bind distinctly different shapes of the hormone. Thus, the focus of the renewal of this 36-year grant is to define shapes of ligands and receptors for la,25(OH)D3 which will describe critical structure-function relationships between ligands (agonists and antagonists) and receptors. Aim 1: Using a 3D molecular model of the VDRnuc and site-directed mutagenesis in the ligand binding domain of VDRnuc, identify the specific amino acids that are critical for: (1.1) optimal ligand binding of the natural hormone 1a,25(OH)D3, and selected conformationally restricted agonist and potential antagonist analogs; and (1.2) formation of a complex between VDRnuc with the GRIP1 coactivator. Aim 2: Determine the shape of analogs of 1a,25(OH)2D3 which optimize their function as agonist or antagonist ligands for (2.1) the VDRnuc, and (2.2) the putative VDRmem. Aim 3 ; For the signal transduction pathway(s) associated with 1a,25(OH)D3-mediated rapid responses: (3.1) Describe upstream pathways of MAP-kinase activation and resultant downstream consequences in the nucleus in human leukemic NB4 cells; (3.2) Determine whether 1a,25(OH)2D3 activates MAP kinase and/or alters gene expression in VDRnucK0 mice; and (3.3) Clone the putative VDRmem/binding protein for 1a,25(OH)2D3 found in chick intestinal basal lateral membranes. The long-term clinical goal is to use rational drug design to chemically synthesize agonist and antagonist analogs of 1 a,25(OH)2D3 to selectively interact with VDRnuc and VDRmem. This may allow specific therapeutic intervention (e.g. type 1 diabetes, leukemia cancer control, psoriasis, immune disorders or osteoporosis) without hypercalcemia.